Large‐scale optical recording of respiration‐modulated neurons in ventrolateral medulla reveals cycle‐to‐cycle variability compatible with multifunctionality

Abstract

Respiration-modulated neurons form a distributed network along the ventral respiratory column (VRC). While anatomically constrained locations are hypothesized to mediate respiration and its coordination with swallow, their interactions are poorly understood. These networks can be recorded optically in the sagittally-sectioned rodent hindbrain (SSRH) preparation. We used neonate (P3-P5) mice, genetically encoded with the Ca2+ indicator GCaMP6F, to record the VRC at high sampling rates, over epochs long enough to robustly characterize the intrinsic cycle-to-cycle variability in both motor output and neuronal onset times. We sought to identify where inspiratory bursts initiated by sorting neuronal onset times relative to ventral root onset. Our findings did not support the hypothesis that respiratory rhythm arises out of the activity of an anatomically compact, dedicated central pattern generating network: earliest neurons were distributed along the VRC, and on a cycle-to-cycle basis, earliest neurons differed. In part, variability in onset times could be accounted for by slow system-level variables: significant correlations between expiratory durations both before and after an inspiratory burst, and neuronal onset times and burst duration were found. We propose that these networks are multifunctional, regulating the coordination of orofacial, swallow, and respiratory behaviors, as well as blood-gas homeostasis. Networks at the mediodorsal edge of the SSRH that carry lung afferent feedback, as well as command networks for orofacial behaviors such as swallow and vocalization are at the surface of SSRH preparation. Thus we can test this hypothesis by evoking associated reflexes and behaviors in vitro using electrical stimulation, and observing respiratory network dynamics.